Defects in base excision repair combined with elevated intracellular dCTP levels dramatically reduce mutation induction in yeast by ethyl methanesulfonate andN-methyl-N′-nitro-N-nitrosoguanidine

Two photorespiratory mutants from Lotus japonicus, namely Ljgln2-1 and Ljgln2-2, deficient in plastidic glutamine synthetase (GLN2), were analysed at the molecular level. Both mutants showed normal levels of Gln2 mRNA, indicating that they were affected post-transcriptionally. Complete sequencing of full-length Gln2 cDNAs revealed the presence of a single point mutation on each mutant, leading to G85R and L278H amino acid replacements, respectively. Different types of experimental approaches, including heterologous expression and complementation tests in Escherichia coli, showed that both GLN2 mutant proteins completely lacked of biosynthetic and transferase enzyme activities. Moreover, it was also shown that while GLN2-1 mutant protein was assembled into a less stable inactive octamer, GLN2-2 mutant protein was unable to acquire a proper quaternary structure and was rapidly degraded. Therefore, the mutations analysed are the first of their type affecting the stability and/or the quaternary structure of the GLN2 enzyme. The kinetic parameters of purified recombinant GLN2 were determined. The enzyme showed positive cooperativity towards ammonium and Mg(2+). Thiol compounds stimulated by twofold the biosynthetic activity but not the transferase activity of recombinant GLN2 and were able to alter the kinetics towards glutamate of the enzyme. Moreover, the biosynthetic activity of recombinant GLN2 was stimulated by more than tenfold by the presence of free Mg(2+).

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Molecular analysis of two mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes

Betti

Arcondéguy

Márquez

2006

Planta

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Involvement of deoxycytidylate deaminase in the response to Sn1-type methylation DNA damage in budding yeast

et al. 2007

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DNA damage-induced mutation: tolerance via translesion synthesis

Kunz

Straffon

Vonarx

2000

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Defects in base excision repair combined with elevated intracellular dCTP levels dramatically reduce mutation induction in yeast by ethyl methanesulfonate andN-methyl-N′-nitro-N-nitrosoguanidine

Cited by 6 publications

References 27 publications

Molecular analysis of two mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes

Molecular analysis of two mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes

Involvement of deoxycytidylate deaminase in the response to Sn1-type methylation DNA damage in budding yeast

DNA damage-induced mutation: tolerance via translesion synthesis

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